www.nature.com/scientificreports OPEN Investigating Chaperonin- Containing TCP-1 subunit 2 as an essential component of the chaperonin complex for tumorigenesis Anne E. Showalter1,6, Ana C. Martini1,6, Daniel Nierenberg1, Kristen Hosang1, Naima Ahmed Fahmi2,3, Priya Gopalan4, Amr S. Khaled5, Wei Zhang 2,3 & Annette R. Khaled1* Chaperonin-containing TCP-1 (CCT or TRiC) is a multi-subunit complex that folds many of the proteins essential for cancer development. CCT is expressed in diverse cancers and could be an ideal therapeutic target if not for the fact that the complex is encoded by eight distinct genes, complicating the development of inhibitors. Few defnitive studies addressed the role of specifc subunits in promoting the chaperonin’s function in cancer. To this end, we investigated the activity of CCT2 (CCTβ) by overexpressing or depleting the subunit in breast epithelial and breast cancer cells. We found that increasing total CCT2 in cells by 1.3-1.8-fold using a lentiviral system, also caused CCT3, CCT4, and CCT5 levels to increase. Likewise, silencing cct2 gene expression by ~50% caused other CCT subunits to decrease. Cells expressing CCT2 were more invasive and had a higher proliferative index. CCT2 depletion in a syngeneic murine model of triple negative breast cancer (TNBC) prevented tumor growth. These results indicate that the CCT2 subunit is integral to the activity of the chaperonin and is needed for tumorigenesis. Hence CCT2 could be a viable target for therapeutic development in breast and other cancers. Te hallmarks of cancer (uncontrolled proliferation, genomic instability, metastasis, etc.) reveal the complex nature of this disease and the challenges faced developing efective therapeutics1,2. Cancer does, however, have an “Achilles heel” and that is its dependency or addiction on major cellular events or processes like transcription, translation, splicing, protein degradation and protein-folding3. In healthy cells, such conserved and essential pro- cesses are rigorously regulated by the proteostasis network (PN) to ensure proteome balance. In order to maintain proteome integrity, the cellular proteome must be synthesized, folded into its native structure, and, when no longer needed, degraded and the amino acids recycled4,5. Chaperones and chaperonins are key players in the PN6. Unlike healthy, non-transformed cells, the PN of cancer cells is taxed to produce proteins involved in sur- vival, angiogenesis, migration, proliferation which are essential for tumor formation, progression and metastasis. Cancer cells have a higher dependency on molecular chaperones and are uniquely challenged due to imbalances caused by chromosomal abnormalities and overexpression of oncogenes, ultimately leading to cellular stress7. As example, inhibitors of Heat Shock Protein 90 (HSP90) showed promising outcomes in the treatment of metastatic breast cancer8. However, despite being in clinical trials since 1998, the success of HSP90 inhibitors in clinical trials remains mixed9–11. Reasons such as dose-limiting toxicity, incomplete inhibition of HSP90, and insuf- cient downregulation of client proteins impeded the clinical use of current HSP90 inhibitors12,13. In recent years, 1Division of Cancer Research, Burnett School of Biomedical Science, College of Medicine, University of Central Florida, Orlando, FL, 32827, USA. 2Department of Computer Science, University of Central Florida, Orlando, FL, 32827, USA. 3Genomics and Bioinformatics Cluster, University of Central Florida, Orlando, FL, 32816, USA. 4Oncology, Department of Internal Medicine, Orlando VA Medical Center, Orlando, FL, 32827, USA. 5Pathology and Laboratory Medicine, Orlando VA Medical Center, Orlando, FL, 32827, USA. 6These authors contributed equally: Anne E. Showalter and Ana C. Martini. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:798 | https://doi.org/10.1038/s41598-020-57602-w 1 www.nature.com/scientificreports/ www.nature.com/scientificreports another class of protein-folding complexes, called chaperonins, gained interest with the recognition that can- cer cells highly express these mediators of protein-folding14–23. Te cytosolic, eukaryotic chaperonin-containing TCP-1 (CCT) is an emerging player in neoplastic transformation with potential for development as a novel diag- nostic marker and therapeutic target. CCT is a type II chaperonin composed of two stacked rings, back-to-back, consisting of eight paralogous subunits (CCT1-8 or CCTα, β, γ, δ, ε, ζ, η, θ)24. Te two rings form a central cavity in which newly synthesized or misfolded proteins can be sequestered and folded in a ATP dependent manner25,26. Previous reports estimated that in eukaryotic cells, CCT facilitates the folding of approximately 10% of newly synthesized proteins, co- and post-translationally27–29, with actin and tubulin being obligate substrates30. In cancer cells, CCT could potentially fold more proteins, with substrates including oncogenic proteins and mediators such as KRAS, STAT3, among others14,20,31–33. Genomic amplifcation of CCT or upregulation of gene expression suggests that CCT could be a marker for oncogenesis34. Previous studies reported that the expression levels of diferent CCT subunits are upregulated in various cancers, such as CCT8 and CCT3 in hepatocellular carcinoma18,22 or glioblastoma35, and CCT2 in prostate, breast, and lung cancers14,15,21. Our lab discovered a CCT inhibitor called CT20p that kills can- cer cells in a CCT-dependent manner. Cancer cells in which CCT was inhibited were resistant to CT20p killing, while cells in which CCT was increased were susceptible14,16. However, the complexity of CCT, with its multiple subunits, and the lack of a full understanding of CCT substrate selectivity in vivo, are some of the challenge that hinder the development of promising therapeutics like CT20p. Te goal of this study is to determine whether the contribution of CCT chaperonin complex to carcinogenesis can be elucidated by focusing on a single subunit, CCT2. We and others showed that CCT2 is increased in breast, prostate, and lung cancer as compared to normal tissues and that CT20p directly interacts with CCT214–16,21. But whether CCT2 is an essential component of the chaperonin’s activity in cancer cells and promotes tumorigenesis is unknown. To address this, we expressed CCT2 as well as silenced cct2 gene expression in epithelial and breast cancer cells. Our study revealed that the levels of CCT2 infuence other CCT subunits and that its expression drives invasiveness and proliferation. CCT2 was needed for tumor growth, indicating that this single subunit could be a viable therapeutic and diagnostic target in cancer. Results CCT is highly expressed in breast cancer and inversely correlates with patient survival. Te CCT complex is encoded by eight diferent genes (tcp1, cct2-8). To determine whether select subunits were upreg- ulated in breast cancer and whether this changed with cancer stage, we performed a comprehensive analysis of the mRNA-sequencing (RNA-seq) data in 1110 breast cancer tumor samples and 116 normal tissue samples from a database compiled by the Akbani lab36. As shown in Fig. 1A, when comparing breast cancer to normal tissue, we found statistically signifcant increases in the majority of the genes expressing CCT subunits. Tis increase in CCT expression was sustained through late stages of the disease. Our previous publication reported that CCT levels were independent of hormone receptor status16, suggesting that the chaperonin levels could be altered in triple negative breast cancer (TNBC). Additional investigation using the same database showed that expression levels for all CCT subunits were signifcantly higher in TNBC tumors compared to normal tissue (Fig. S1A). Breast cancer patient survival data obtained from the Kaplan-Meier Plotter (KMPlotter) dataset showed that patient survival inversely correlated with CCT levels in tumor tissue (Fig. S1B), suggesting that high expression of the chaperonin associates with poor patient prognosis. While these data indicate that CCT could be a good therapeutic target whose inhibition positively impacts patient outcomes, targeting eight genes for inhibition is a challenge difcult to surmount. It would be ideal to target a single CCT subunit by demonstrating its essential role in promoting the chaperonin’s activity. To this end, we found that CCT2 protein levels, but not CCT3 as example, varied among breast cancer cell lines, with highest levels of CCT2 in TNBC cells and lowest in luminal A types and non-transformed breast epithelial cells (Fig. S1C,D; CCT3 shown as a representative of other subunits). Te importance of CCT2 in breast cancer was further examined with patient data from Te Cancer Genome Atlas (TCGA), showing that only genomic alterations in CCT2 resulted in statistically signifcant diferences in patient survival (Fig. 1B,C). Patients with genomic alterations in CCT2 (mainly gene amplifcation or increased mRNA) died almost 70 months sooner than patients without changes in CCT2. Tese fndings led us to investigate the role of CCT2 in cancer progression using CCT2 overexpressed or depleted cells. CCT2 expression promotes invasiveness and increased levels of other CCT subunits. In order to evaluate the efects of increased CCT2 in cells, we generated two versions of a plasmid, FLAG-CCT2 and CCT2-FLAG, with the FLAG tag on the N-terminal or C-terminal of CCT2 respectively (Fig. S2A) and used a third-generation lentiviral system for gene delivery. To determine transfection efciency, the